Extracellular Vesicles Contribute to Mixed-Fungal Species Competition during Biofilm Initiation

ABSTRACT Extracellular vesicles commonly modulate interactions among cellular communities. Recent studies demonstrate that biofilm maturation features, including matrix production, drug resistance, and dispersion, require the delivery of a core protein and carbohydrate vesicle cargo in Candida species. The function of the vesicle cargo for these advanced-phase biofilm characteristics appears to be conserved across Candida species. Mixed-species interactions in mature biofilms indicate that vesicle cargo serves a cooperative role in preserving the community. Here, we define the function of biofilm-associated vesicles for biofilm initiation both within and among five species across the Candida genus. We found similar vesicle cargo functions for several conserved proteins across species, based on the behavior of mutants. Repletion of the adhesion environment with wild-type vesicles returned the community phenotype toward reference levels in intraspecies experiments. However, cross-species vesicle complementation did not restore the wild-type biology and in fact drove the phenotype in the opposite direction for most cross-species interactions. Further study of mixed-species biofilm adhesion and exogenous wild-type vesicle administration similarly demonstrated competitive interactions. Our studies indicate that similar vesicle cargoes contribute to biofilm initiation. However, vesicles from disparate species serve an interference competitive role in mixed-Candida species scenarios.


Biofilm adhesion assay 11
Ninety-six-well flat-bottom polystyrene plates were used to assess biofilm adhesion (2). Fungal 12 cell inocula (10 6 cells/ml) were prepared out of overnight yeast cultures in YPD at 30°C, followed 13 by dilution in RPMI-MOPS based on count numbers with an automated Countess™ II cell counter 14 (Invitrogen). One hundred µl of yeast cells per well were seeded. Plates were incubated 90 min 15 at 37°C, the growth medium was then removed and non-adherent cells were gently washed out 16 with PBS. The density of adhered fungal cells was determined by the XTT assay as described 17 above. Adhesion capacity of biofilms was calculated using the change in absorbance compared 18 to that of controls. 19

Exogenous extracellular vesicle addback assays and functional network construction 20
Biological impact of exogenous extracellular vesicles on Candida biofilm adhesion was 21 determined in 96-well plates. Both reference wild type and CHT3 null deletion mutants were used 22 in this assay. Fungal cell inocula (10 6 cells/ml) were prepared out of overnight yeast cultures in 23 YPD at 30°C, followed by dilution in RPMI-MOPS based on count numbers with the automated 24 Countess™ II cell counter (Invitrogen). One hundred µl of yeast cells per well were seeded. 25 Extracellular vesicles isolated from all five tested Candida species were used in combinations in 26 biofilms of all five Candida species at normalized concentrations ranging between 1×10 4 and 27 3×10 6 particles/ml. Exogenous extracellular vesicles were applied during biofilm inoculation. 28 Biofilms growth in cultures with and without exogenous extracellular vesicles was evaluated by 29 the XTT assay (2). The obtained phenotypic outcomes were organized into a visual Candida 30 biofilm phenotypic network using the Cytoscape platform (3). 31

Dual Candida species adhesion assay 32
Adhesion of dual Candida species biofilms was assessed in 96-well plates. Fungal cell inocula 33 (10 6 cells/ml) were prepared out of overnight yeast cultures in YPD at 30°C, followed by dilution 34 in RPMI-MOPS based on count numbers with the automated Countess™ II cell counter 35 (Invitrogen). We initially tested two different seeding options (either 5×10 4 cells/species/well or 36 1×10 5 cells/species/well), but we concluded that the first concentration choice inculated into a 37 limited well surface area (~0.32 cm 2 ) provided more accurate results. Thus, a total of one hundred 38 µl of yeast cells (50 µl of each species) per well were seeded. Cells were mixed right before 39 seeding and plates were then incubated for 90 min at 37°C, the growth medium was removed, 40 and non-adherent cells were gently wash out with PBS. Next, 100 µl of 20 mM NaOH was added 41 to each well and fungal biofilm cells were lysed for 20 min at 100°C (2). To determine the 42 abundance of individual Candida species, quantitative real-time PCR (qRT-PCR) was performed 43 as follows: 20 µl reactions were set up in triplicate in 96 well plates, using 0.5 µl of isolated DNA 44 aliquots per reaction. Real time primer-probe sets sequences (IDT) used for the assay are listed 45 in Table S2. Reactions were run using the PrimeTime ® Gene Expression Master Mix (IDT) per 46 manufacturer's recommendations. Standard curves were generated with genomic DNA isolated 47 from 10 8 cells from tested strains isolated with the MasterPure™ DNA Yeast Isolation Kit 48 (Lucigen), and serially diluted in PCR-grade water. Cell concentration for standard curves was 49 quantified prior to isolation with the Countess II Automated Cell Counter (Invitrogen). Real time 50 cycles were as follows: one 3 min step at 95°C, followed by 40 cycles of 95°C for 15 s, and then 51 60 s at 60°C with read step. qRT-PCR was run on the BioRAD CFX96 Real Time PCR system 52 (BioRad), and analysis was performed using CFX Maestro 2.3 software (BioRad). The obtained 53 phenotypic outcomes were then organized into a visual Candida biofilm adhesion network using 54 the Cytoscape platform (3). 55

Extracellular vesicle isolation and analysis 56
Candida biofilms were grown using a large-scale rolling bottle biofilm model system. Culture 57 media were carefully decanted from the polystyrene bottles after 24 and 48 h of incubation at 58 37°C. Culture supernatants were filter sterilized and concentrated down to about 25 ml using a 59 Vivaflow 200 unit (Sartorius AG) equipped with a Hydrosart 30 kDa cut-off membrane. Samples 60 were centrifuged to remove smaller cellular debris particulates first at 10,000×g for 1 h at 4°C. 61 The pellet was discarded, and the resulting supernatant was centrifuged again at 100,000×g for 62 1.5 h at 4°C. Next, the supernatant was discarded, and the pellet was then washed in 5 ml of PBS 63 and re-centrifuged at 100,000×g for 1 h at 4°C. The collected extracellular vesicles were next 64 polished by flash size-exclusion chromatography on a qEV/35 nm column (Izon Science), filter 65 sterilized and stored until further use at 4°C (4). 66 Exosomes were quantified using nanoparticle tracking analysis (NTA). EV samples were diluted 67 in PBS to a final volume of 1 ml and pretested to obtain an ideal 30-100 particles per frame rate 68 using a NanoSight NS300 system coupled with an autosampler (Malvern). The following settings 69 were applied: camera level was increased to 16 and camera gain to 2 until tested images were 70 optimized and nanoparticles were distinctly visible without exceeding particle signal saturation. 71 Each measurement consisted of five 1-min videos with a delay of 5 s between sample introduction 72 and the start of the first measurement. For detection threshold analysis the counts were limited to 73 10-100 red crosses and no more than 5-7 blue crosses. Acquired data were analyzed using the 74 NanoSight Software NTA 3.4 Build 3.4.003. At least 1000 events in total was tracked per sample 75 in order to minimize data skewing based on single large particles (5). 76

Assessment of extracellular vesicle production in biofilms during adhesion 77
Quantitative analysis of EVs produced during adhesion of Candida biofilms was determined at in 78 96-well plates. Fungal cell inocula (10 6 cells/ml) were prepared out of overnight yeast cultures in 79 YPD at 30°C, followed by dilution in RPMI-MOPS based on count numbers with the automated 80 Countess™ II cell counter (Invitrogen). One hundred µl of yeast cells per well were seeded and 81 incubated at 37°C. Supernatant samples were collected after 90 min of incubation, filter sterilized 82 and subjected to NTA-based extracellular vesicle analysis as described above (Fig S1). 83